Isocratic Reverse-Phase High-Performance Liquid Chromatographic Assay for Simultaneous Estimation of Emtricitabine, Tenofovir Disoproxil Fumarate, and Rilpivirine in Fixed-Dose Combination Tablets

Research Article

Abstract

An isocratic reverse-phase high-performance liquid chromatographic (RP-HPLC) method was validated for the concurrent quantitative determination of emtricitabine, tenofovir disoproxil fumarate, and rilpivirine in triple-combination pharmaceutical dosage forms. Chromatographic separation was achieved on a Hypersil BDS C18 column (250 times 4.6 mm ID, 5 µm particle size) utilizing a mobile phase consisting of potassium dihydrogen phosphate buffer (adjusted to pH 4.0 with orthophosphoric acid) and acetonitrile (35:65 v/v) at a flow rate of 1.0 mL/min with ultraviolet detection at 280 nm. Chromatographic elution yielded sharp, symmetrical peaks with mean retention times of 2.692 min, 4.402 min, and 5.725 min for emtricitabine, tenofovir disoproxil fumarate, and rilpivirine, respectively, within a total run time of 10 min. Beer-Lambert linearity was confirmed over concentration ranges of 50-300 µg/mL for emtricitabine, 75-450 µg/mL for tenofovir disoproxil fumarate, and 6.25-37.5 µg/mL for rilpivirine, each exhibiting linear regression correlation coefficients (R²) exceeding 0.999. Method precision demonstrated relative standard deviations below 1.0% across repeatability, intra-day, and inter-day evaluations. Standard addition recovery studies yielded mean percentages of 99.79-100.72% for emtricitabine, 99.47-100.03% for tenofovir disoproxil fumarate, and 99.58-99.98% for rilpivirine. Limits of detection were 0.0128 µg/mL, 0.380 µg/mL, and 0.0016 µg/mL, while limits of quantitation were 0.039 µg/mL, 1.154 µg/mL, and 0.0049 µg/mL, respectively. Deliberate modifications in operational parameters confirmed analytical robustness without altering critical chromatographic attributes. The established protocol provides reliable throughput and reproducibility suitable for routine pharmaceutical quality control.